The plasmalemmal serotonin transporter (SERT) terminates serotonergic neurotransmission and thus plays a critical role in shaping the duration and magnitude of synaptic signaling. It works by coupling preexisting ion gradients to the thermodynamically unfavorable movement of serotonin from the synapse to neuronal and glial cytoplasms. This molecular machine has garnered significant clinical attention because its dysfunction has been implicated in multiple debilitating neuropsychiatric diseases such as depression, autism, obsessive- compulsive disorder, and generalized anxiety. Furthermore, it is the target of numerous psychoactive agents such as antidepressants, cocaine, and the amphetamine derivative ecstasy. Despite such clinical and pharmacological significance, atomic-level detail into the mechanism of substrate translocation, inhibition, and regulation has remained elusive. Two goals of this proposal include 1) solving the structure of a SERT in complex with substrates, ions, and inhibitors; and 2) probing the conformational dynamics of these interactions via hydrogen-deuterium exchange mass spectrometry (HDX-MS). Structure-based hypotheses will subsequently be tested with a combination of site-directed mutagenesis, cysteine crosslinking, transient and steady-state flux kinetics, dissociation/association binding kinetics, X-ray crystallography, and HDX-MS. Success will permit further investigation into specific antagonist binding sites and perhaps into the molecular basis for drug resistance, thereby paving the way for rational drug design efforts. When eventually coupled with in vivo work beyond the scope of this proposal, SERT structure/function studies may also shed light on the molecular underpinnings of disease-associated polymorphisms. Achieving any of one of these objectives would likely have significant impact in both the laboratory and the clinic.